Polymethine compounds and their use as fluorescent labels

Abstract

The present disclosure relates to new compounds and their use as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

The invention claimed is: 
     
       1. A compound of the formula (I), or a mesomeric form thereof: 
       
         
           
           
               
               
           
         
         wherein mCat+ or mAn− is an organic or inorganic positively/negatively charged counterion; 
         m is an integer 0-3; 
         p is an integer 1 or 2; 
         q is an integer 1-5; 
         alk is a chain of 1-5 carbon atoms optionally containing one or more double or triple bonds; 
         Y is S or O; 
         Z is OH; 
         n is 0 or 1; 
         X is OH or O − , or an amide or ester conjugate thereof; 
         each of Ra 1  and Ra 2  is independently H, SO 3   − , sulfonamide, halogen, or a further ring fused to an adjacent carbon atom; and 
         each of Rc 1  and Rc 2  is independently alkyl or substituted alkyl, wherein at least one of Ra 1  or Ra 2  is SO 3   − , or Ra 1  or Ra 2  is a further ring fused to an adjacent carbon atom, the further ring having an SO 3   − , or Rc 1  or Rc 2  is an alkyl sulfonic acid group. 
       
     
     
       2. The compound according to  claim 1 , wherein (alk) is (CH 2 ) 3 . 
     
     
       3. The compound according to  claim 1 , wherein q is 5. 
     
     
       4. The compound according to  claim 1 , wherein each of Ra 1  and Ra 2  is H or SO 3   − . 
     
     
       5. The compound according to  claim 1 , wherein Ra 1  or Ra 2  is a further ring fused to an adjacent carbon atom. 
     
     
       6. The compound according to  claim 1 , wherein Rc 1  or Rc 2  is methyl, ethyl, propyl or —(CH 2 ) t SO 3   −  where t is 1-6. 
     
     
       7. The compound according to  claim 6 , wherein either Rc 1  or Rc 2  is —(CH 2 ) 4 —SO 3   − . 
     
     
       8. The compound according to  claim 1 , wherein Y is O. 
     
     
       9. The compound according  claim 1 , wherein n is 1. 
     
     
       10. The compound according to  claim 1 , wherein either indole moiety of formula (I) is part of a structure: 
       
         
           
           
               
               
           
         
         wherein Rd is H, alkyl, substituted alkyl, aryl, substituted aryl, halogen, carboxy, sulfonamide, or sulfonic acid; and 
         wherein Rc 1  is alkyl or substituted alkyl. 
       
     
     
       11. The compound according to  claim 1 , which is represented by formula (VIIIa), or a mesomeric form thereof: 
       
         
           
           
               
               
           
         
         wherein 
         r is an integer 1-5. 
       
     
     
       12. The compound according to  claim 1 , which is represented by formula (VIId), or a mesomeric form thereof: 
       
         
           
           
               
               
           
         
         wherein 
         r is an integer 1-5; and 
         t is an integer 1-6. 
       
     
     
       13. A nucleotide or oligonucleotide labelled with a compound according to  claim 1 . 
     
     
       14. A nucleotide or oligonucleotide labelled with a compound according to  claim 12 , wherein the compound is attached to the nucleotide or oligonucleotide via an amide linkage formed from the C(═O)—X moiety. 
     
     
       15. The nucleotide or oligonucleotide according to  claim 13 , wherein the compound is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base of the nucleotide or oligonucleotide through a linker moiety. 
     
     
       16. The nucleotide or oligonucleotide according to  claim 13 , further comprising a 3′ OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide or oligonucleotide. 
     
     
       17. A kit comprising two or more nucleotides wherein at least one nucleotide is a labelled nucleotide according to  claim 13 . 
     
     
       18. The kit according to  claim 17 , wherein two of the labelled nucleotides are excited using a single laser. 
     
     
       19. The kit according to  claim 18 , wherein a first of four nucleotides is a labelled nucleotide according to  claim 13 , and the second, third, and fourth nucleotides are each labelled with a different compound, wherein each compound has a distinct absorbance maximum and each of the compounds is distinguishable from the other three compounds. 
     
     
       20. The kit according to  claim 17 , comprising four nucleotides wherein a first of the four nucleotides is a labelled nucleotide according to  claim 13 , a second of the four nucleotides is a labelled nucleotide according to  claim 13 , a third nucleotide carries a third label and a fourth nucleotide is unlabelled (dark). 
     
     
       21. The kit according to  claim 17  comprising four nucleotides wherein a first of the four nucleotides is a labelled nucleotide according to  claim 13 , a second of the four nucleotides is a labelled nucleotide according to  claim 13 , a third nucleotide carries a mixture of two labels and a fourth nucleotide is unlabelled (dark). 
     
     
       22. Use of a nucleotide or oligonucleotide according to  claim 13  in sequencing, expression analysis, hybridisation analysis, genetic analysis, RNA analysis or protein binding assays. 
     
     
       23. Use according to  claim 22 , on an automated sequencing instrument wherein said automated sequencing instrument comprises two lasers operating at different wavelengths. 
     
     
       24. A method of synthesising a compound according to  claim 1 , utilising one of the following starting materials: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         or a salt thereof; 
         wherein Ra 1  is H, SO 3   − , sulfonamide, halogen, or a further ring fused to an adjacent carbon atom; 
         Rc 1  is alkyl or substituted alkyl; 
         r is an integer 1-5; 
         Ar is an aromatic group; and 
         R is an alkyl group. 
       
     
     
       25. A method of synthesising a compound according to  claim 1 , utilising the following starting material: 
       
         
           
           
               
               
           
         
       
     
     
       26. The compound according to  claim 11 , wherein X is OH and q is 5. 
     
     
       27. The compound according to  claim 12 , wherein X is OH and q is 5. 
     
     
       28. The compound according to  claim 1 , selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         and mesomeric forms thereof. 
       
     
     
       29. The nucleotide or oligonucleotide according to  claim 14 , comprising the compound moiety: 
       
         
           
           
               
               
           
         
         or a mesomeric form thereof. 
       
     
     
       30. A nucleotide or oligonucleotide labelled with a compound according to  claim 11 , wherein the compound is attached to the nucleotide or oligonucleotide via an amide linkage formed from the C(═O)—X moiety. 
     
     
       31. The nucleotide or oligonucleotide according to  claim 30 , comprising the compound moiety: 
       
         
           
           
               
               
           
         
         or a mesomeric form thereof.